Assessment method

ABSTRACT

A method of assessing the viability of a cell comprises incubating the cell in a culture medium. The culture medium includes a plurality of amino acids and the change in concentration in the medium of at least one amino acid is determined.

FIELD OF THE INVENTION

[0001] This invention relates to a method of assessing the viability ofa cell.

BACKGROUND OF THE INVENTION

[0002] Amino acids have been shown to improve the development ofpre-implantation embryos in vitro in a variety of species, such as themouse (Gardner and Lane 1993), rat (Zhang and Armstrong 1990; Miyoshi etal. 1995), sheep (Gardner et al. 1994) and cow (Takahashi and First1992; Rosenkrans and First 1994; Keskintepe et al. 1995); Lane andGardner (1994) reported that Eagle's essential amino acids increased theinner cell mass (ICM) cell numbers in murine embryos cultured from thezygote stage.

[0003] Embryos in vivo, derive exogenous amino acids from oviducal anduterine fluids. A total of 20 free amino acids have been detected inbovine oviducal fluid (Stanke et al 1974), and 25 have been detected inbovine uterine fluid (Fahning et al. 1967). Moore and Bondioli (1993)found glycine and alanine to be the two most predominant amino acids inbovine oviducal fluid and that supplementation with these amino acidsenhanced bovine embryo development in the presence of oviducal cells.Suh et al. (1995) reported that significantly more bovine zygotescultured in CR2 medium with glycine reached the blastocyst stage. Riegerand Loskutoff (1994) have shown that glutamine and glycine are consumedby denuded bovine oocytes, and that glutamine is taken up during earlypre-implantation development (Rieger et al. 1992).

[0004] Although studies in this area have concentrated on administeringsingle or pairs of radiolabelled amino acids, embryos within the femaletract will be exposed to a mixture of amino acids (Leese 1988). Lamb andLeese (1994) measured the consumption of a physiological mixture of 20amino acids by murine blastocysts, and found that 9 were depletedsignificantly.

[0005] The fate of amino acids in bovine embryos has been investigatedby Frei et al. (1989) who measured the rate of incorporation ofradiolabelled methionine into protein. They found a quantitativedecrease in the rate of protein synthesis between the zygote and 8-cellstage, followed by a progressive increase from this point to theblastocyst stage. The quantitative increase in amino acid utilisationobserved around these stages of development could be related to theinitiation of transcription of the bovine embryonic genome which occursat the 8-16-cell stage of development (Telford et al. 1990).

[0006] Amino acids have also been shown to improve the development ofbovine zygotes fertilised in vitro to morulae and blastocysts and toincrease total cell numbers at the blastocyst stage (Takahashi and First1992; Rosenkrans and First 1994; Keskintepe et al. 1995). It is notclear how exogenously-administered amino acids assist embryo developmentin vitro; some, such as glutamine, may act as energy sources (Rieger andGuay 1988; Rieger 1992), others may increase the pool size of endogenousamino acids and thereby stimulate de novo protein synthesis (Zhang andArmstrong 1990). Van Winkle and Dickinson (1995) have shown that thereare significant differences between the amino acid content of murineembryos that develop in vitro and those that develop in vivo.

[0007] Partridge and Leese (1996) investigated bovine embryos which hadbeen cultured with 19 amino acids at concentrations routinely used tosupplement the medium synthetic oviduct fluid (SOF; Tervit et al. 1972).Groups of embryos fertilised in vitro from the putative zygote stage tothe blastocyst stage, and blastocysts freshly flushed from the uterus onDay 7 after fertilisation (derived in vivo) were studied. Depletionrates for 17 of the amino acids were measured over a 12-h period withindividual amino acids detected by high performance liquidchromatography (HPLC) following fluorimetric derivatisation.

[0008] Partridge and Leese (1996) found glutamine depletion at theputative zygote stage (0.76±0.05 pmol zygote⁻¹ h⁻¹) and at the 4-cellstage (0.94±0.1 pmol embryo⁻¹ h⁻¹). However, a greater depletion ofglutamine was not observed at the blastocyst stage, in contrast to theresults of Rieger et al. (1992) who measured the uptake of radiolabelledglutamine given as a single amino acid substrate in B2 medium by bovineembryos.

[0009] With regard to amino acid depletion, a most intriguing result ofPartridge and Leese (1996) was the depletion of threonine in significantamounts at all stages of development in vitro as well as by theblastocyst derived in vitro. The fate of threonine is unknown, but itcould act as an energy substrate, by entering the Krebs cycle asacetyl-Coenzyme A (CoA) or succinyl-CoA.

[0010] Alanine was produced in significant amounts by all stages ofembryos produced in vitro and by embryos derived in vivo. Van Winkle andDickinson (1995) hypothesised that alanine could act as a route forembryos to sequester waste nitrogen since very high concentrations werefound in murine blastocysts grown in vitro. In addition, Gardner andLane (1993) have shown that ammonia toxicity is a potential problem formouse embryos grown in vitro. The large increase in external alanineconcentration observed during the culture of bovine embryos produced invitro and derived in vivo in the present study leads us to suggest thatalanine may indeed be formed by the embryo to prevent the build-up oftoxic ammonium ions.

[0011] The inclusion of amino acids in human pre-implantation culturemedium has become more prevalent since the advent of blastocyst transferand the requirement for increased embryo development beyond the 4- to8-cell stage.

[0012] In spite of this, there is still little knowledge regarding whichamino acids are actually utilised by the embryo at various stages ofdevelopment.

[0013] Current methods for in-vitro embryo production include in-vitrofertilisation and intra cytoplasmic sperm injection (ICSI). Embryoproduction may also follow the techniques of cryopreservation and embryobiopsy.

[0014] Understanding the way in which embryos modify an amino acidmixture may provide a clue to understanding why the embryo produced invitro is less robust than its in vivo counterpart. These problems areparticularly apparent in human in vitro fertilisation (IVF) programmeswhereby the average rate of success in the UK is currently about 17% or1 in 6.

[0015] A typical human IVF programme involves the administration ofovarian egg production and releasing hormones to the woman. These eggsare collected and inseminated with sperm to generate about ten embryos.Up to three (in the UK) of the fertilised embryos will then betransferred back to the woman and if the programme is successful, atleast one will implant itself in the womb and continue to develop.

[0016] In an effort to reduce hormone administration, eggs may becollected at the earlier stages of oogenesis. Subsequent maturation ofthe eggs occurs in-vitro. Following insemination of the in-vitro maturedeggs, up to three fertilised embryos are then transferred back to thewoman for implantation.

[0017] The method of intra cytoplasmic sperm injection is nowincreasingly used for fertilisation. Subsequent to the administration ofovarian egg production and releasing hormones, eggs, surrounded bycumulus cells, are released. The protective layer obscures the egg andmust be removed to reveal an egg which is then subjected to a system ofvisual grading before sperm injection is carried out.

[0018] To date there exists no method by which embryos or eggs withincreased development potential can be effectively and reliablyselected, although glucose consumption and lactate production have beenused in the mouse for this purpose. Comparative studies of physiologicalparameters such as glucose, pyruvate, or oxygen consumption in arrestingand healthy embryos or eggs have failed to provide a solution to theproblem. Current methods rely on morphological selection whereby embryosand eggs are subjected to a grading system.

[0019] Because of the great uncertainty in the determination of the mostviable embyros and eggs, the need to transfer more than one embryo backinto the mother for implantation after artificial insemination becomesapparent. This procedure compensates for the likelihood that one or moreof the embryos may fail to develop and serves to heighten the limitedchances of success.

[0020] Increasing the reliability of the egg or embryo selection willhave important ramifications upon the IVF programme as a whole wherebythe most viable embryo can be selected and transferred for subsequentimplantation. The transfer of a single viable embryo guards against thepossibility of multiple births which carries the risk of premature birthand perinatal problems.

[0021] It should be understood that any test does not need to be 100%accurate or reliable but should simply provide a non invasive method forconsistent indication as to the viability of a single egg or embryo.

[0022] A suitable test should involve a selection period which is asshort as possible so that transfer of the embryo and implantation cantake place as soon as possible after in vitro fertilisation. Thisminimises any risks which might be associated with prolonged exposure ofthe developing embryo to the artificial culture conditions. A shorterselection period is also beneficial from an economic point of viewbecause the costs of an otherwise labour and resource intensiveoperation can be minimised.

[0023] Considerable research interest is also focused on the generationof embryos by nuclear transfer (NT). Such embryos are made by injectinga nucleus from a donor cell (karyoplast) into an enucleated egg(ooplast) and then using an electric pulse to trigger embryodevelopment. A variety of karyoplasts have been used for nucleartransfer including stem cells, which are derived from the inner cellmass of the blastocyst and which are the precursor cells for alltisssues of the body. However, embryo-derived stem cells (ES cells) haveonly been conclusively isolated from the mouse and the human and thereis an intensive search for methods to produce them in other speciesincluding the domestic species. In the case of ‘Dolly’ the karyoplastwas a somatic (adult) mammary gland cell.

[0024] The generation of embryos by nuclear transfer, especially fromstem cells, is the preferred route towards the production of transgenicanimals and for cell ‘therapeutic cloning’—the production of new cellsand tissues to replace those which have become diseased or ceased tofunction properly. However, current methods for the production andidentification of karyoplasts, stem cells, stem cell precursors andviable nuclear transfer embryos are laborious and time consuming.

[0025] There is a need for a biochemical marker(s) which would simplifythe identification of a cell such as a gamete (which may be at any stageof development), an embryo (which may be made by nuclear transfer), akaryoplast, a putative stem cell population, a stem cell precursorpopulation or a stem cell population.

[0026] As used herein the term ‘egg’ refers to an egg at any stage ofoogenesis and includes in-vitro matured eggs.

STATEMENTS OF THE INVENTION

[0027] According to the present invention there is provided a method ofassessing the viability of a cell comprising incubating the cell in aculture medium including a plurality of amino acids and determining thechange in concentration in the medium of at least one amino acid.

[0028] The term cell is used in its broadest sense and refers to agamete (which may be at any stage of development), an embryo (which maybe made by nuclear transfer), a karyoplast, a putative stem cellpopulation, a stem cell precursor population or a stem cell population.

[0029] The term viability is used in its broadest sense to encompass,amongst other things, the development of an embryo to the blastocyststage, successful implantation of an embryo and pre-implantationscreening methods.

[0030] Preferably the method of the present invention further comprisesthe steps of selecting the cell if the change meets a predeterminedcriterion.

[0031] Preferably an embryo which is selected for further development isintroduced into the uterine tract of an organism and implants in theuterine wall. An egg which is selected for further development isfertilised to produce an embryo which is introduced into the uterinetract of an organism and implants in the uterine wall. In one embodimentof the invention the egg is an in-vitro matured egg. A karyoplast,putative stem cell population, stem cell precursor population, stem cellpopulation or embryo made by nuclear transfer which is selected forfurther development may be used in the production of a transgenicorganism with desirable qualities such as disease resistance, high leanmass and capacity to produce human medical products in its milk.

[0032] Preferably the culture medium comprises Earle's Balanced SaltSolution (EBSS) supplemented with glucose, L-lactate, pyruvate and aphysiological mixture of amino acids.

[0033] Preferably the concentrations of glucose, L-lactate and pyruvaterange from 0.5 mM to 1.5 mM, 4 mM to 6 mM and 0.37 mM to 0.57 mMrespectively. More preferably the concentrations of glucose, L-lactateand pyruvate are 1 mM, 5 mM, and 0.47 mM.

[0034] Preferably an embryo (which may be made by nuclear transfer),egg, karyoplast, putative stem cell population, stem cell precursorpopulation or stem cell population is cultured in approximately 4 μldrops of culture medium. The concentration of amino acids in the spentmedium is measured using HPLC, preferably followed by derivatisationwith o-phthaldialdehyde, and an amino acid consumption and productionprofile is generated.

[0035] In order to achieve accurate dilution of the microlitre samplesfor use in HPLC, an internal standard is introduced into the medium.Preferably the internal standard is D-alpha-aminobutyric acid.

[0036] The present invention also provides for a diagnostic kitincluding means for incubating a cell in a culture medium and means fordetermining the change in concentration in the medium of at least oneamino acid. The diagnostic kit generates an amino acid profile showingconsumption or production of amino acids in the culture medium in whichthe test cell is incubated. Preferably the diagnostic kit allows for acomparison of the amino acid profile of the incubated cell withpredetermined ‘finger-print’ amino acid profiles for arresting andhealthy cells of a particular organism of study. Accordingly, the aminoacid profiles are used as a selection marker in the selection of themost viable cells.

[0037] The amino acid consumption or production profile can be used toverify the presence of a putative stem cell population, stem cellprecursor population or stem cell population which will be characterisedby a particular amino acid profile. The putative stem cell population,stem cell precursor population or stem cell population can then be usedin genetic manipulation to produce transgenic organisms. The method ofthe present invention represents an enormous advantage over currenttechniques by providing a simple biochemical marker for stem cellidentification and selection.

[0038] Using the method of the present invention, selection of a cell ispreferably achieved in 24 hours or less after transfer of the cell intothe culture medium. More preferably selection is achieved in 10 hours orless after transfer of the cell into the culture medium. Most preferablyselection is achieved in 6 hours or less.

[0039] In one embodiment of the invention the ‘fingerprint’ amino acidprofile is used as a whole as a selection marker to select a viable cellfor a particular species. In a separate embodiment of the invention,selection of the most viable cell is based upon a smaller group of aminoacids, typically comprising one to five amino acids, whose consumptionor production profile is indicative of a healthy cell for that species.

[0040] The method of the invention may be used for a variety oforganisms including humans, cows, pigs, sheep and any other domesticanimals. The amino acids used for a selection marker may include any ora plurality of amino acids. In one embodiment of the invention, themethod is used for humans and the amino acids used for a selectionmarker include any or a combination of the amino acids alanine,arginine, aspartic acid, glutamic acid, glutamine, glycine, leucine,lysine, tryptohan, valine. In one embodiment of the invention, the aminoacid used for a selection marker is alanine.

DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention will now be described by way of exampleonly and with reference to the accompanying drawings wherein:

[0042]FIG. 1 shows the mean amino acid consumption or production profile(pmol/embryo/h) for 27 human embryos which arrested development at the8-cell stage. Amino acids which were significantly consumed or producedare marked with an asterix.

[0043]FIG. 2 shows the mean amino acid consumption or production profile(pmol/embryo/h) for 22 human embryos at the compacting 8-cell stagewhich developed to the blastocyst stage. Amino acids which weresignificantly consumed or produced are marked with an asterix.

[0044]FIG. 3 is a superimposition of the data from FIGS. 1 and 2 andcompares the amino acid consumption or production (pmol/embryo/h) forhuman embryos which arrested development at the 8-cell stage (shadedbars) and human embryos which developed to the blastocyst stage(unshaded bars). T-tests were used to compare the consumption orproduction data for each amino acid for each group of arresting anddeveloping embryos. A p-value of less than 0.05 was consideredsignificant. Data for the amino acids marked with the superscript a, b,c or d (alanine, asparagine, glycine and lysine respectively) aresignificantly different for the two sets of tests.

[0045]FIG. 4 is a superimposition of data to compare amino acidconsumption or production (pmol/embryo/h) for human embryos from day 2to day 3 post fertilisation which arrested development at the 8-cellstage (shaded bars) and human embryos from day 2 to day 3 postfertilisation which developed to the blastocyst stage (unshaded bars).

[0046]FIG. 5 shows the sum of utilisation of five amino acids indeveloping and arresting human embryos from day 2 to day 3 postfertilisation.

[0047]FIG. 6 is a superimposition of data to compare amino acidconsumption or production (pmol/embryo/h) for ICSI embryos that resultedin pregnancy (unshaded bars) and that failed to result in pregnancy(shaded bars) from day 1 to day 2 of development.

[0048]FIG. 7 shows the sum of Gly, Ala and Trp appearance for ICSIembryos that resulted and failed to result in pregnancy, from day 1 today 2 of development.

[0049] In initial experiments, spare in vitro fertilised human embryoswere individually placed in 4 μl drops of culture medium until theyreached the blastocyst stage. Embryos were cultured individually tomimic the conditions which are encountered by an embryo developing invivo. The culture medium comprised 4 μl drops of EBSS supplemented with1 mM glucose, 5 mM L-lactate, 0.47 mM pyruvate and a physiologicalmixture of 20 amino acids. The individual concentrations of the aminoacids were in the range 0.005 mM to 1.0 mM.

[0050] During incubation, the concentration of 18 amino acids in thespent medium was simultaneously measured using high performance liquidchromatography (HPLC) following derivatisation with o-phthaldialdehyde.The results were used to compile amino acid consumption and productionprofiles as illustrated in FIGS. 1 to 3.

[0051] In order to achieve accurate dilution of microlitre samples foruse in HPLC, an internal standard in the form of the non metabolisableamino acid; D-alpha-aminobutyric acid, was introduced into the medium ata concentration of 1 in 49 parts per volume. This internal standardallowed the minute differences that occur in resting embryos, and whichwould otherwise be lost in the ‘back-ground noise’ to be picked up. TheHPLC peak attributable to the marker can be easily distinguished and wasused to calculate the correct dilution.

[0052] As shown in FIG. 2, the only amino acids that were significantlydepleted from the medium during the compacting 8-cell to the morulastage were serine, arginine, isoleucine and leucine. Alanine, aspartate,glutamate and tryptophan were significantly produced. Alanine appearanceincreased from 10.87±1.61 pmol/embryo/h for embryos cultured between thecompacting 8-cell to the morula stage compared to 13.90±1.23pmol/embryo/h for embryos cultured from the morula to blastocyst stage.

[0053] Embryos that arrested at the compacting 8-cell stage producedsignificantly more alanine (17.72±1.44 pmol/embryo/h; p=0.0028) thandeveloping embryos of the same stage (FIGS. 1 to 3). Glutamate was alsosignificantly produced over the morula to blastocyst transition.

[0054] Although it is known that an embryo will cause changes to theamino acid concentration of a culture medium in which it is incubated,the significant differences in the changes brought about by healthy andarresting embryos have previously gone unnoticed. It should beunderstood that the results of the embryo experiments are highlyindicative of those which would be expected for an egg, karyoplast, stemcell, stem cell precursor or embryo made by nuclear transfer subjectedto the same incubation conditions. Accordingly, the change inconcentration of at least one amino acid in the culture medium can beused to give an indication as to the viability of an embryo, egg,karyoplast, stem cell, stem cell precursor or embryo made by nucleartransfer. This represents a major advance in the selection of embryos oreggs for subsequent implantation and the selection of karyoplasts, stemcells, stem cell precursors or embryos made by nuclear transfer forgenetic manipulation.

[0055] The increased production of alanine by arresting embryos forexample is surprising and counter-intuitive. If alanine is used as aroute to sequester waste nitrogen and is formed by the embryo to preventthe build-up of toxic ammonium ions, it might be expected that thehealthiest embryos would be most metabolically active and produce higherquantities of alanine as compared with arresting embryos. Surprisingly,the results show that healthiest embryos are metabolically ‘quieter’ andthat embryos which fail to develop turnover comparatively more proteinwith the amino groups being exported into the culture medium as alanine.

[0056] The results indicate that the human pre-implantation embryo isable to use amino acids selectively at different stages of developmentand that the appearance of alanine in the medium may be used as apotential marker of the viability of an embryo, egg, karyoplast, stemcell, stem cell precursor or embryo made by nuclear transfer.

[0057] The method of the present invention could be used inpre-implantation screening for diseases such as phenylketonuria (PKU),cystic fibrosis and other such genetic or chromosomal abnormalities.

[0058] All babies in the UK are currently tested for PKU a few daysafter birth by measuring the phenylalanine level in the blood usingchromatography or a bacterial growth test. It is likely that thoseembryos or eggs which are predestined to PKU are characterised by adifferent amino acid profile compared with that produced by a healthyembryo or egg. Accordingly the method of the present invention carrieshuge potential for future genetic screening programs.

[0059] The method of the present invention may also have immense valuein sex determination whereby embryos of different sex may becharacterised by a certain amino acid profile.

[0060] It should be understood that the method of the invention has widereaching applications and is not limited to the use of a human embryo,egg, karyoplast, stem cell, stem cell precursor or embryo made bynuclear transfer. A typical method in cattle breeding is theadministration of egg inducing hormones to a cow of high genetic meritfollowed by natural insemination leading to the production of about 6 to8 embryos in the uterus of the animal. The embryos are then ‘flushed’from the cow and transferred singly to lower grade animals forsubsequent development. Due to the risks of improper flushing, thistechnique is unethical for use in man.

[0061] Alternatively, egg production in a valuable cow is hormonallyinduced and following retrieval (ovum pick-up), the eggs areartificially inseminated using high quality sperm and cultured to theblastocyst stage before subsequent transferral back to a recipient.Cattle embryos may also be generated from oocytes derived from abattoirovaries.

[0062] Such cattle breeding programmes are of multi-national concern andany selection method by which the most viable eggs or embryos fortransfer can be selected represents a major industrial advance.

[0063] Other animals to which this technology may be applicable includesheep, pigs, all domestic animals and rare and threatened species. Thecloning technology used in production of ‘Dolly’ was met by low successrates with 276 previously failed attempts. The ‘Dolly’ programme alsoinvolved considerable expenditure of research effort in the attempt togenerate appropriate sheep cells to produce transgenic animals.

[0064] The method of the present invention now provides a rationalapproach to the selection of the most viable cell for use in subsequentembryo or egg transfer and to the selection of karyoplasts, putativestem cell populations, stem cell precursor populations, stem cellpopulations and embryos made by nuclear transfer. It represents a majoradvance in farm animal improvement techniques which involvemicro-manipulation, nuclear transfer and the addition of geneticconstructs.

[0065] It should be understood that the amino acid profile for a cell ofa particular organism may be highly discrete; differing in detail as towhich particular amino acids are consumed and produced and also whichparticular amino acids are consumed and produced in arresting anddeveloping cells when compared with that profile generated for a cell ofa different organism. Although the results suggest that alanine may beused as a potential marker for the viability of human cells, furtherinvestigations may reveal that other amino acids are more suitablemarkers for other species. Indeed, further investigation of the humanembryo reveal that additional amino acids besides alanine are alsosuitable for use as a selection marker.

[0066] The initial study was based upon human embryos from day 3 to day4 post insemination. As shown in FIG. 3, lysine is produced in arrestingembryos and consumed in healthy embryos. This might have selectionpotential. Similarly the results for asparagine and glycine aresignificant. It was suspected that as more tests are carried out, thesignificant differences between amino acid consumption and productionfor healthy and arresting embryos may increase.

[0067] Studies were extended to human embryos from day 2 to day 3 postinsemination. Earlier assessment was considered advantageous since it ispreferable to minimise the exposure of the developing embryo toartificial culture conditions as far as possible. The results of theseexperiments are illustrated in FIGS. 4 and 5.

[0068] Subsequent studies involved the collection of pregnancy data.ICSI embryos were analysed from day 1 to day 2 of development, beforesubsequent transfer into patients. The choice of ICSI embryos versus IVFembryos was due to the fact that the cumulus of an ICSI embryo has beenremoved, thus facilitating assessment. Now the focus was not so much theidentification of healthy, pre-implantation embryos but whether theembryo would implant within the mother (i.e., a move towards theclinical side).

[0069] ICSI embryos were incubated in culture medium comprising EBSSsupplemented with 1 mM glucose, 5 mM L-lactate, 0.47 mM pyruvate and aphysiological mixture of 20 amino acids. The individual concentrationsof the amino acids were in the range 0.005 mM to 1.0 mM.

[0070] The results from this line of the investigation were particularlyencouraging and provide corroborative evidence for the efficacy of theassessment method.

[0071] The method of the present invention allows for the generation of‘finger-print’ amino acid profiles which are characteristic for aparticular species and which can be used in selecting the most viablecell for that species.

[0072] Because it is not known which embryo will develop to term,typically two or three embryos are transferred back into the motherafter artificial insemination. The method of the present inventionreduces the likely of multiple pregnancy by providing a biochemical testfor the selection of a single embryo. This is considerably more accuratethan morphological selection methods.

1. A method of assessing the viability of a cell wherein said cell is agamete, an embryo, a karyoplast, a putative stem cell population, a stemcell precursor population or a stem cell population, said methodcomprising incubating the cell in a culture medium including a pluralityof amino acids and determining the change in concentration in the mediumof at least one amino acid.
 2. A method according to claim 1 furthercomprising the steps of selecting said cell for further development ifthe change meets a predetermined criterion.
 3. A method according toclaim 2 wherein the cell is an embryo and the embryo is introduced intothe uterine tract of an organism.
 4. A method according to claim 2wherein the cell is an egg and the egg is fertilised to produce anembryo which is introduced into the uterine tract of an organism.
 5. Amethod according to claim 2 wherein the cell which is selected forfurther development is used in the production of a transgenic organismor for therapeutic replacement of diseased or damaged cells in tissues.6. A method according to any preceding claim wherein the culture mediumcomprises Earle's Balanced Salt Solution supplemented with glucose,L-lactate, pyruvate and a physiological mixture of amino acids.
 7. Amethod according to claim 6 wherein the concentrations of glucose,L-lactate and pyruvate range from 0.5 mM to 1.5 mM, 4 mM to 6 mM and0.37 mM to 0.57 mM respectively and the concentrations of the individualamino acids range from 0.005 mM to 1.0 mM.
 8. A method according toclaim 7 wherein the concentrations of glucose, L-lactate and pyruvateare 1 mM, 5 mM, and 0.47 mM.
 9. A method according to any precedingclaim wherein a gamete, embryo, karyoplast, putative stem cellpopulation, stem cell precursor population or stem cell population iscultured in culture medium.
 10. A method according to any precedingclaim wherein the concentration of amino acids in the spent medium ismeasured using HPLC followed by derivatisation with o-phthaldialdehyde.11. A method according to claim 10 wherein an internal standard isintroduced into the sample medium in order to achieve accurate dilutionof the microlitre samples for use in HPLC.
 12. A method according toclaim 11 wherein the internal standard is D-alpha-aminobutyric acid. 13.A method according to any preceding claim wherein an amino acidconsumption or production profile is generated.
 14. A method accordingto claim 13 wherein said amino acid consumption or production profile isused as a whole as a selection marker in asssessing the viability of acell.
 15. A method according to claim 13 wherein selection of the mostviable cell is based upon a group of amino acids, typically comprisingtwo to seven amino acids, whose consumption or production profile isindicative of a healthy, developing cell for that species.
 16. A methodaccording to claim 13 wherein selection of the most viable cell is basedupon a single amino acid, whose consumption or production profile isindicative of a healthy developing cell for that species.
 17. A methodaccording to any preceding claim wherein assessment of viability of acell is achieved in 24 hours or less after transfer of the cell into theculture medium.
 18. A method according to claim 17 wherein assessment ofviability of a cell is achieved in 10 hours or less after transfer ofthe cell into the culture medium.
 19. A method according to claim 18wherein assessment of viability of a cell is achieved in 6 hours or lessafter transfer of the cell into the culture medium.
 20. A methodaccording to any preceding claim wherein the cell is derived from anyorganism including humans, cows, pigs, sheep, any domestic animal or arare and threatened species.
 21. A method according to claim 20 whereinthe method is used for humans.
 22. A method according to claim 21wherein the amino acid used for a selection marker includes one or acombination of alanine, cysteine, aspartic acid, glutamic acid,phenylalanine, glycine, histidine, isoleucine, lysine, leucine,methionine, asparagine, proline, glutamine, arginine, serine, threonine,valine, tryptophan or tyrosine.
 23. A method according to claim 22wherein the amino acid used for a selection marker is alanine.
 24. Atransgenic non-human animal produced in accordance with the method ofclaim
 5. 25. A diagnostic kit including means for incubating a cell in aculture medium including a plurality of amino acids and means fordetermining the change in concentration in the medium of at least oneamino acid.
 26. A diagnostic kit according to claim 25 wherein thediagnostic kit generates a profile of consumption or production for eachamino acid.
 27. A diagnostic kit according to claim 25 or claim 26wherein the kit provides means for selecting a cell for furtherdevelopment if the change meets a predetermined criterion.